Analytical description of adolescent binge drinking patients

Background Binge drinking is a widespread health compromising behavior among adolescents and young adults, leading to significant health problems, injuries and mortality. However, data on alcohol consumption is often unreliable, as it is mainly based on self-reporting surveys. In this five-year study (2014–2019) at the University Children’s Hospital Zurich, we analyzed blood samples from adolescent binge drinking patients to investigate blood alcohol concentrations (BACs), co-ingestion of drugs, assess compliance between self-reported and measured substance use, and test for genetic components of innate alcohol tolerance. Furthermore, hair analysis was performed to retrospectively access drug exposure and to evaluate the potential of hair analysis to assess binge drinking. Methods In a prospective, single-center study, patients with alcohol intoxications aged 16 years and younger were included. Blood and hair samples were analyzed by sensitive liquid chromatography – tandem mass spectrometry drug analysis. HTTLPR genotyping was performed with PCR and fragment analysis. Results Among 72 cases, 72 blood and 13 hair samples were analyzed. BACs ranged from 0.08–3.20‰ (mean 1.63‰, median 1.60‰), while a mean concentration of 3.64 pg/mg hair (median 3.0 pg/mg) of the alcohol marker ethyl glucuronide (EtG) was detected in eleven hair samples, providing no evidence of chronic excessive drinking. In 47% of the cases, co-ingested drugs were qualitatively detected next to ethanol, but only 9% of the detected drugs had blood concentrations classified as pharmacologically active. Cannabis consumption (22%) and stimulant intake (16%) were the most frequently observed drugs. Compliance between patients’ statements and measured substances matched well. Although we investigated the genetic contribution to innate alcohol tolerance via the 5-HTTLPR polymorphism, the diverse genetic background of the cohort and small sample size did not allow any conclusions to be drawn. Conclusion Almost half of our binge drinking patients tested positive for other substances, primarily cannabis. We anticipate that our study enhances understanding of consumption behavior of young people and encourage continued efforts to address the harmful effects of binge drinking and co-occurring substance use. Supplementary Information The online version contains supplementary material available at 10.1186/s12887-023-04325-2.

A and B. Subsequently, three separate analyses were conducted: untargeted screening analysis, confirmative, quantitative analysis of a multitude of drugs, and identification and quantification of cannabis, similar to a previous study [1].

Whole Blood (qualitative) screening by LC-MS/MS
Extracted blood samples were analyzed using an untargeted data acquisition approach with a Toxtyper® LC-MS/MS system.The system was composed of an UltiMate 3000 LC system controlled by Chromeleon 6.80 software (Thermo Fischer Scientific, Reinach, Switzerland) and an Amazon Speed TM ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a standard electrospray ionization (ESI)-source.Separation was achieved using an Acclaim TM RSLC 120 C18 column (2.1 x 100 mm, 2.2 µm, Thermo Fisher Scientific) at a flow rate of 0.5 mL/min and an injection volume of 5 µL.The gradient elution method was programmed as follows: 0-1 min 1% B; from 1-4 min to 21% B, from 4-8 min to 29% B, 8-13 min increase to 64% B and till 15 min to 99% B; hold for 2 min; decrease to starting conditions and re-equilibration for 2 min.The MS was operated in auto-MSn-mode (Ultrascan, m/z 70-800, 32500 m/z per second).A positive electrospray ionization mode with the following source parameters was applied, capillary voltage: 4500 V; end plate offset: 500 V; nebulizer gas: 7.3 psi; dry gas: 4.0 L/min; and dry temperature: 180°C.The trap ion charge control was set to 300000 and the maximal accumulation time was 100 ms.The collision-induced fragmentation (CID) threshold was 10000 units, the amplitude was set to 0.8 V. MS/MS spectra were triggered for the highest intensity ion, followed by exclusion after one occurrence.Data were automatically matched to the Maurer/Wissenbach/Weber database [2], and reports were generated using Data Analysis 4.2 and Compass Open Access software (Bruker Daltonik GmbH).

Quantification of drugs in blood
Quantification of positive results from the blood screening analysis was performed by LC-MS/MS using a targeted in-house multianalyte method.The method covered 82 drugs or drug metabolites from various drug classes such as stimulants, opioids, benzodiazepines, antidepressants, antipsychotics, antitussives, and antihistamines.Limits of quantifications (LOQs) were as follows: 2.4 ng/mL, 6 ng/mL, 2.4 ng/mL, 2.4 ng/mL, 1.6 ng/mL, 8.0 ng/mL, 8.0 ng/mL, 20 ng/mL, 4.0 ng/mL, 2.0 ng/mL, and 16 ng/mL for amphetamine, benzoylecgonine, cocaine, dextromethorphan, diphenhydramine, doxylamine, ketamine, lorazepam, MDMA, methylphenidate, and quetiapine, respectively.Briefly, measurements were carried out on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific) coupled to a Sciex 5500 QTrap linear ion trap quadrupole mass spectrometer (Sciex, Darmstadt, Germany).The LC settings were as follows: Phenomenex (Aschaffenburg, Germany) Kinetex PS C18 (2.6 µm, 100 A, 100x2.1 mm), gradient elution with 10 mM ammonium formate buffer in water containing 0.1 % (v/v) formic acid (pH 3.5, eluent A), and acetonitrile containing 0.1 % (v/v) formic acid (eluent B).Positive electrospray ionization (ESI) was used, and the MS was operated in scheduled multiple reaction monitoring (MRM) mode using two transitions for each analyte.MS was controlled by Analyst® 1.6.2software (Sciex).Quantitation was performed using MultiQuant® 2.1.1 software (Sciex).The method was fully validated according to national and international guidelines [3][4][5][6].Quantification was performed on the peak area ratios of drug to IS against an eight-point calibration covering an extended therapeutic range for prescription drugs and ranges typically observed in forensic toxicology for drugs of abuse.
Each analysis batch was controlled by additional randomized measurements of three quality control samples.

Quantitative analysis for cannabinoids in blood
Given the undetectability of cannabinoids in the screening analysis, all blood samples were subjected to targeted cannabis analysis on tetrahydrocannabinol (THC), cannabidiol (CBD) and the THC metabolites hydroxy-THC (THC-OH) and THC carboxylic acid (THC-COOH).LOQs were as follows: 0.5 ng/mL, 0.2 ng/mL, 0.5 ng/mL, and 5 ng/mL for THC, CBD, THC-OH, and THC-CCOH, respectively.Sample extracts were analyzed on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific) coupled to a Sciex 5500 QTrap linear ion trap quadrupole mass spectrometer (Sciex).The LC settings were as follows: Phenomenex (Aschaffenburg, Germany) Kinetex C18 (2.6 µm, 100A, 50 x 2.1mm), gradient elution with 5 mM ammonium formate buffer in water containing 0.1 % (v/v) formic acid (eluent A) and methanol (eluent B); injection volume 10 µL.MS mode, QC, and data evaluation were performed as described above for the drug quantification.

Analysis of drugs, medication and ethylglucuronide in hair samples
Hair samples were first analyzed for drugs and medication to assess past drug exposure using a multianalyte approach by Scholz et al. [7].Hair samples were washed with 15 mL water, 10 mL acetone, and 10 mL hexane.Each washing step was performed for 2 min, with slight shaking.The samples were dried and chopped into snippets using scissors.Approximately 20 mg of the snippets were exactly weighed into Eppendorf tubes and pulverized using a Retsch ball mill (Type MM 400, Retsch GmbH & Co. KG, Haan, Germany) by shaking in the presence of one tungsten carbide ball (Ø 7 mm, 3 g, Retsch) at 30 Hz for 10 min.After pulverization, 1.4 mL of methanol and 0.1 mL of an internal standard (IS) solution were added to each sample [7].The Eppendorf tubes, together with the remaining tungsten carbide balls, were again placed in the ball mill and shaken at 10 Hz for 90 min.After centrifugation at 9000 rpm for 25 min, the supernatant was collected and dried at 35 °C under nitrogen.For the second microextraction step, 1 mL of 1-mM aqueous ammonium formate containing 0.1% formic acid/methanol (1:1, v/v) was added to the remaining hair powder, followed by shaking at 10 Hz for 90 min in a ball mill.The resulting supernatant was combined with the dried supernatant from the first step and dried at 35 °C under nitrogen.The dried residues were reconstituted in 150 μL methanol, vortexed, and 350 μL of a solution of 2-mM aqueous ammonium formate was added.The solutions were transferred to liquid chromatography (LC) vials.The LC-MS-MS system consisted of a Shimadzu Prominence high performance liquid chromatography system (Shimadzu, Duisburg, Germany) and a QTrap 5500 mass spectrometer (Sciex) using electrospray ionization (ESI) operating in positive mode.Separation was achieved using a Kinetex® F5 column (100 mm × 2.1 mm, 100 Å, 2.6 µm, Phenomenex) coupled with SecurityGuard™ ULTRA Cartridges ultra-high performance liquid chromatography (UHPLC) F5 (2.1 mm ID).Mobile phase A (water containing ammonium formate [1 mM] and formic acid [0.1%]) and mobile phase B (acetonitrile containing ammonium formate [1 mM] and formic acid [1 mM]) were used.A post-column spray of methanol was applied at a flow rate of 0.04 mL/min in order to support the ionization process, especially at the beginning of the gradient.The flow rate was set at 0.6 mL/min, the gradient was programmed as follows: 0.01 to 1.5 min, 3% eluent B; 1.5 to 9 min increasing to 60% eluent B; 9 to 10 min increasing to 95% eluent B; 10 to 11 min, 95% eluent B; 11 to 11.1 min decreasing to 3% eluent B, 11.1 to 12 min starting conditions (3% eluent B).The column oven temperature was set at 40°C.The dead time (t0) was about 0.3 min (0.19-mL void volume of the column).
In a second step hair samples that had enough material (> 5 mg) were analyzed for ethylglucuronide.EtG analysis (Limit of detection (LOD) 1 pg/mg and limit of quantification (LOQ) 2 pg/mg)) was performed following the protocol described by Binz et al. [8].Briefly, the selected hair segment was washed twice with 15 ml of H2O, followed by 15 ml acetone in a Sarstedt tube for 2 min.The hair samples were subsequently dried at room temperature and cut into small pieces of a few millimeters.Approximately 20 mg of hair were filled into an Eppendorf tube (2 ml) and pulverized with a single tungsten carbide ball in a ball mill.After pulverization, 1000 pg d5-EtG as internal standard and 1.5 ml of water were added, and samples were extracted twice for 15 min.Solid-phase extraction (SPE) was then performed using Waters Oasis1MAX cartridges.The cartridges were equilibrated using 2 ml of methanol and 2 ml of water.The supernatant from the samples was transferred to the cartridges, followed by 1 ml of water/ammonia (5%) solution and a washing step with 2 ml of methanol.The columns were then dried under vacuum for 5 min.Finally, the EtG was eluted with 2 ml of 2% formic acid in methanol.The eluate was evaporated under nitrogen at 35 °C using a metal heating block.The LC-MS system consisted of a Shimadzu Prominence XR high-pressure liquid-chromatography (HPLC) system coupled to a Sciex QTrap 5500 linear ion trap quadrupole mass spectrometer (Sciex).EtG was separated employing a Hypercarb TM PGC column (100 x 2.1 mm, 3 µm) with a guard column (10 x 2.1 mm, 3 µm) at 40 °C (Thermo Fisher Scientific).The mobile phase consisted of water with 1% ammonium (A) and acetonitrile (B), at a flow rate of 0.4 ml/min.The gradient was as follows: 0-5% B for 0-5 min, 5-90% B from 5 to 8 min, isocratic 90% B from 8 to 11 min, 90-0% B from 11t o 13 min, and an equilibration step of 1 min.The post column solvent addition of methanol was performed at 0.2 ml/min.The 5500 ion trap instrument was operated in negative ion mode with an ion-spray voltage of 4500 V, a source temperature of 600 °C, nitrogen at 20 psi as curtain gas, and nebulation and heating gases at 80 psi and 85 psi, respectively.The analytes were detected in multiple reaction monitoring mode (MRM), monitoring three transitions for EtG (221/75, 221/85 and 221/55) and two transitions for d5-EtG, (226/75 and 226/85).Analysis of the collected data was carried out using the Analyst software (version 1.6.2,Sciex).The collision-associated dissociation gas was set to ''high''.All source parameters were optimized under LC conditions and the electrical parameters were optimized by direct infusion.